Pseudoazurin binds at a single site on cytochrome c peroxidase from Paracoccus pantotrophus with a K(d) of 16.4 microM at 25 degrees C, pH 6.0, in an endothermic reaction that is driven by a large entropy change. Sedimentation velocity experiments confirmed the presence of a single site, although results at higher pseudoazurin concentrations are complicated by the dimerization of the protein. Microcalorimetry, ultracentrifugation, and (1)H NMR spectroscopy studies in which cytochrome c550, pseudoazurin, and cytochrome c peroxidase were all present could be modeled using a competitive binding algorithm. Molecular docking simulation of the binding of pseudoazurin to the peroxidase in combination with the chemical shift perturbation pattern for pseudoazurin in the presence of the peroxidase revealed a group of solutions that were situated close to the electron-transferring heme with Cu-Fe distances of about 14 A. This is consistent with the results of (1)H NMR spectroscopy, which showed that pseudoazurin binds closely enough to the electron-transferring heme of the peroxidase to perturb its set of heme methyl resonances. We conclude that cytochrome c550 and pseudoazurin bind at the same site on the cytochrome c peroxidase and that the pair of electrons required to restore the enzyme to its active state after turnover are delivered one-by-one to the electron-transferring heme.

The crystal structure of the aldehyde oxido-reductase (Mop) from the sulfate reducing anaerobic Gram-negative bacterium Desulfovibrio gigas has been determined at 2.25 A resolution by multiple isomorphous replacement and refined. The protein, a homodimer of 907 amino acid residues subunits, is a member of the xanthine oxidase family. The protein contains a molybdopterin cofactor (Mo-co) and two different [2Fe-2S] centers. It is folded into four domains of which the first two bind the iron sulfur centers and the last two are involved in Mo-co binding. Mo-co is a molybdenum molybdopterin cytosine dinucleotide. Molybdopterin forms a tricyclic system with the pterin bicycle annealed to a pyran ring. The molybdopterin dinucleotide is deeply buried in the protein. The cis-dithiolene group of the pyran ring binds the molybdenum, which is coordinated by three more (oxygen) ligands.

The kinetic properties of the electron-transfer process between reduced Desulfovibrio vulgaris cytochrome c3 and D. vulgaris flavodoxin have been studied by anaerobic stopped-flow techniques. Anaerobic titrations of reduced cytochrome c3 with oxidized flavodoxin show a stoichiometry of 4 mol of flavodoxin required to oxidize the tetraheme cytochrome. Flavodoxin neutral semiquinone and oxidized cytochrome c3 are the only observable products of the reaction. At pH 7.5, the four-electron-transfer reaction is biphasic. Both the rapid and the slow phases exhibit limiting rates as the flavodoxin concentration is increased with respective rates of 73.4 and 18.5 s-1 and respective Kd values of 65.9 +/- 9.4 microM and 54.5 +/- 13 microM. A biphasic electron-transfer rate is observed when the ionic strength is increased to 100 mM KCl; however, the observed rate is no longer saturable, and relative second-order rate constants of 5.3 x 10(5) and 8.5 x 10(4) M-1 s-1 are calculated. The magnitude of the rapid phase of electron transfer diminishes with the level of heme reduction when varying reduced levels of the cytochrome are mixed with oxidized flavodoxin. No rapid phase is observed when 0.66e(-)-reduced cytochrome c3 reacts with an approximately 25-fold molar excess of flavodoxin. At pH 6.0, the electron-transfer reaction is monophasic with a limiting rate of 42 +/- 1.4 s-1 and a Kd value of approximately 8 microM. Increasing the ionic strength of the pH 6.0 solution to 100 microM KCl results in a biphasic reaction with relative second-order rate constants of 5.3 x 10(5) and 1.1 x 10(4) M-1 s-1. Azotobacter vinelandii flavodoxin reacts with reduced D. vulgaris cytochrome c3 in a slow, monophasic manner with limiting rate of electron transfer of 1.2 +/- 0.06 s-1 and a Kd value of 80.9 +/- 10.7 microM. These results are discussed in terms of two equilibrium conformational states for the cytochrome which are dependent on the pH of the medium and the level of heme reduction [Catarino et al. (1991) Eur. J. Biochem. 207, 1107-1113].

The kinetic properties of the electron-transfer process between reduced Desulfovibrio vulgaris cytochrome c(3) and D. vulgaris flavodoxin have been studied by anaerobic stopped-flow techniques. Anaerobic titrations of reduced cytochrome c(3) with oxidized flavodoxin show a stoichiometry of 4 mol of flavodoxin required to oxidize the tetraheme cytochrome. Flavodoxin neutral semiquinone and oxidized cytochrome c(3) are the only observable products of the reaction. At pH 7.5, the four-electron-transfer reaction is biphasic. Both the rapid and the slow phases exhibit limiting rates as the flavodoxin concentration is increased with respective rates of 73.4 and 18.5 s(-1) and respective K-d values of 65.9 +/- 9.4 mu M and 54.5 +/- 13 CIM. A biphasic electron-transfer rate is observed when the ionic strength is increased to 100 mM KCl; however, the observed rate is no longer saturable, and relative second-order rate constants of 5.3 X 10(5) and 8.5 x 10(4) M(-1) s(-1) are calculated. The magnitude of the rapid phase of electron transfer diminishes with the level of heme reduction when varying reduced levels of the cytochrome are mixed with oxidized flavodoxin. No rapid phase is observed when 0.66e(-)-reduced cytochrome c(3) reacts with an similar to 25-fold molar excess of flavodoxin. At pH 6.0, the electron-transfer reaction is monophasic with a limiting rate of 42 +/- 1.4 s(-1) and a Kd value of similar to 8 mu M. Increasing the ionic strength of the pH 6.0 solution to 100 mu M KCl results in a biphasic reaction with relative second-order rate constants of 5.3 x 10(5) and 1.1 x 10(4) M(-1) s(-1) Azotobacter vinelandii flavodoxin reacts with reduced D. vulgaris cytochrome cs in a slow, monophasic manner with limiting rate of electron transfer of 1.2 +/- 0.06 s(-1) and a K-d value of 80.9 +/- 10.7 mu M. These results are discussed in terms of two equilibrium conformational states for the cytochrome which are dependent on the pH of the medium and the level of heme reduction [Catarino et al. (1991) Eur. J. Biochem. 207, 1107-1113].

Ni and Se x-ray absorption spectroscopic studies of the [NiFeSe]hydrogenases from Desulfovibrio baculatus are described. The Ni site geometry is pseudo-octahedral with a coordinating ligand composition of 3-4 (N,O) at 2.06 A, 1-2 (S,Cl) at 2.17 A, and 1 Se at 2.44 A. The Se coordination environment consists of 1 C at 2.0 A and a heavy scatterer M (M = Ni or Fe) at approximately 2.4 A. These results are interpreted in terms of a selenocysteine residue coordinated to the Ni site. The possible role of the Ni-Se site in the catalytic activation of H2 is discussed.